Bacterial Respiratory Pathogens Research Articles

Evaluation of Bacterial Co-infections of the Respiratory Tract in COVID-19 Patients

Introduction: The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2. The co-evolution of viral and bacterial respiratory pathogens has created an environment in which a viral infection allows concurrent or secondary bacterial co-infections. Aims: This study was carried out to isolate the different respiratory bacterial pathogens from COVID patients and evaluate their antibiotic sensitivity pattern. Methods: Hospital based descriptive cross sectional study was conducted over a period of between 2nd Jestha to 5th Shrawan 2078 (16th May to 20th July 2021) in Nepalgunj Medical college, Kohalpur during the second wave of COVID-19 in Nepal. The sputum was inoculated onto different culture plates such as Blood Agar (BA) and MacConkey Agar (MA) and incubated at 37 oC. After overnight growth and in Gram staining, different Gram’s positive and negative bacteria were observed. Antibiotic sensitivity test was performed by Kirby-Bauer Disk Diffusion method. Results: Out of 100 sputum samples, 44% positive bacterial growths, majority isolates were Klebsiella spp and Staphylococcus aureus. Overall, the pathogens were mostly sensitive to meropenem followed by colistin, piperacilin/tazobactam respectively. Conclusion: Bacterial co-infection is common in hospitalized patients with SARS-CoV-2.

Respiratory Tract Pathogens in the COVID-19 Era: Data from a Pediatric Emergency Department

Abstract Objective The frequency of coinfections in pediatric Coronavirus disease 2019 (COVID-19) cases and their impact on the clinical course are not fully understood. We aimed to investigate the viral and bacterial respiratory pathogens in children admitted to the pediatric emergency department (PED), their clinical course, and the presence of coinfections during the early months of the COVID-19 pandemic. Methods Clinical, laboratory and radiological findings, viral and bacterial pathogens detected by multiplex polymerase chain reaction (PCR) tests in nasopharyngeal swabs, clinical course, and treatments of all children who were tested for severe acute respiratory coronavirus 2 (SARS-CoV-2) at the PED between March 16 and May 15, 2020, were recorded. SARS-CoV-2 PCR-positive and negative groups were compared. Results Out of 570 patients tested for SARS-CoV-2 during the study period, 43 were found positive (7.5%). Non-SARS-CoV-2 viral pathogens were more common in the SARS-CoV-2 PCR-negative group than the SARS-CoV-2 PCR-positive group (13.2%, n = 68 versus 4.7%, n = 2), but this result was not statistically significant. Leukocyte, neutrophil, lymphocyte, and platelet counts were lower in SARS-CoV-2 PCR-positive group. Bacterial panel positivity was significantly higher in the SARS-CoV-2 PCR-positive group compared with the SARS-CoV-2 PCR-negative group (52%, n = 12 versus 28%, n = 91; p < 0.05). The presence of coinfection did not alter the course of therapy in SARS-CoV-2 PCR-positive cases. Conclusion While viral coinfections were rare, bacterial panel positivity was common in children with COVID-19, but this had not influenced management decisions. The limitations of the tests should be kept in mind while interpreting the results.

COVID-19 Pandemic Impact on Respiratory Infectious Diseases in Primary Care Practice in Children.

Background: The coronavirus disease 2019 (COVID-19) pandemic led to day care and school closures and children staying home for several months. When they gradually returned, aggressive regulations were implemented in New York State to reduce viral transmission.Method: An ongoing prospective study occurring in the Rochester, NY region, focused on early childhood respiratory infectious diseases, afforded an opportunity to assess the impact of the pandemic on the incidence of these illnesses in a primary care outpatient setting. Physician-diagnosed, medically attended infection visits were assessed in two child cohorts, age 6–36 months old: from March 15 to December 31, 2020 (the pandemic period) compared to the same months in 2019 (prepandemic). Nasopharyngeal colonization by potential otopathogens during healthy/well-child and acute otitis media (AOM) visits was evaluated.Results: One hundred and forty-four children were included in the pandemic cohort and 215 in the prepandemic cohort. The pandemic cohort of children experienced 1.8-fold less frequent infectious disease visits during the pandemic (p < 0.0001). Specifically, visits for AOM were 3.7-fold lower (p < 0.0001), viral upper respiratory infections (URI) 3.8-fold lower (p < 0.0001), croup 27.5-fold lower (p < 0.0001), and bronchiolitis 7.4-fold lower (p = 0.04) than the prepandemic cohort. Streptococcus pneumoniae (p = 0.03), Haemophilus influenzae (p < 0.0001), and Moraxella catarrhalis (p < 0.0001) nasopharyngeal colonization occurred less frequently among children during the pandemic.Conclusion: In primary care pediatric practice, during the first 9 months of the COVID-19 pandemic, significant decreases in the frequency of multiple respiratory infections and nasopharyngeal colonization by potential bacterial respiratory pathogens occurred in children age 6–36 months old.

Fatal Calf Pneumonia Outbreaks in Italian Dairy Herds Involving Mycoplasma bovis and Other Agents of BRD Complex.

Mycoplasma bovis is increasingly recognized worldwide as an important cause of disease with major welfare and production impairments on cattle rearing. Although it was detected in veal calves and beef cattle, little is known on the infection impact and on its temporal morbidity pattern in Italian dairy herds. Thus, this study aimed to investigate the involvement of M. bovis on fatal calf pneumonia outbreaks that occurred during 2009–2019 in 64 Italian dairy farms. Furthermore, a deeper diagnostic workup of concurrent infection with other viral and bacterial respiratory pathogens was assessed. Out of the investigated fatal pneumonia cases, M. bovis was frequently detected (animal prevalence, 16.16%; 95%CI, 11.82–21.33; herd prevalence, 26.56; 95%CI, 16.29–39.08) either as the single agent of the disease in more than half of the positive samples (20/37) or in concurrent infections with Histophilus somni (9/37, 24.3%), Mannheimia haemolytica (6/37, 16.621%), Trueperella pyogenes (1/37, 2.70%), Pasteurella multocida (1/37, 2.70%), bovine respiratory syncytial virus (5/37, 13.51%), and bovine viral diarrhea virus (2/37, 5.55%). Based on time-series analysis, M. bovis was recorded in the area since 2009 with outbreaks displaying a clear morbidity seasonal pattern with peaks in April (43.21%) and in September (13.51%). This might be due to the stressing conditions during spring and late summer periods. Results of this study highlight that M. bovis infection warrants consideration, and control measures are needed given its involvement in lethal pneumonia outbreaks in dairy herds from an extended area.

Cleavage of E-cadherin by porcine respiratory bacterial pathogens facilitates airway epithelial barrier disruption and bacterial paracellular transmigration

ABSTRACT Airway epithelial cells are the first line of defense against respiratory pathogens. Porcine bacterial pathogens, such as Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Glaesserella (Haemophilus) parasuis, and Pasteurella multocida, breach this barrier to lead to local or systematic infections. Here, we demonstrated that respiratory bacterial pathogen infection disrupted the airway epithelial intercellular junction protein, E-cadherin, thus contributing to impaired epithelial cell integrity. E-cadherin knocking-out in newborn pig tracheal cells via CRISPR/Cas9 editing technology confirmed that E-cadherin was sufficient to suppress the paracellular transmigration of these porcine respiratory bacterial pathogens, including G. parasuis, A. pleuropneumoniae, P. multocida, and B. bronchiseptica. The E-cadherin ectodomain cleavage by these pathogens was probably attributed to bacterial HtrA/DegQ protease, but not host HtrA1, MMP7 and ADAM10, and the prominent proteolytic activity was further confirmed by a serine-to-alanine substitution mutation in the active center of HtrA/DegQ protein. Moreover, deletion of the htrA gene in G. parasuis led to severe defects in E-cadherin ectodomain cleavage, cell adherence and paracellular transmigration in vitro, as well as bacterial breaking through the tracheal epithelial cells, systemic invasion and dissemination in vivo. This common pathogenic mechanism shared by other porcine respiratory bacterial pathogens explains how these bacterial pathogens destroy the airway epithelial cell barriers and proliferate in respiratory mucosal surface or other systemic tissues.

Influenza A as a Common Viral Cause of Complex Febrile Seizures

Abstract Objective The most common childhood convulsive disorder happens to be febrile seizure (FS), which is an important health problem leading to economic burden and parental anxiety. Further investigation into the etiological causes of FS will guide us for appropriate measures during the follow-up period. The aim of study was to identify the percentage of viral and bacterial pathogens in the etiological causes of children with FS, and also if there is any difference between simple and complex FSs. Methods This prospective study randomly enrolled 100 pediatric patients with FS between January 2017 and July 2017. Nasopharyngeal swabs were obtained from all children at presentation. The respiratory panel was performed with a multiplex real-time polymerase chain reaction method to detect the 21 most common viruses. A complete blood count, absolute neutrophil count, absolute lymphocyte count, C-reactive protein, erythrocyte sedimentation rate, procalcitonin, blood culture, throat culture, urine analyses, urinary culture, and stool tests analysis were performed in all the patients. Results During the study period, at least one virus was detected in 87% of patients. Bacterial agents were detected in only 13% of patients. Coinfections of the viruses and bacterial pathogens were found in 24% of patients. The most frequently detected virus was influenza A (Inf A) (18%), followed by rhinovirus (12%). Coinfections of the viruses and bacterial pathogens, mixed viral infections, and Inf A were common in children who experienced complex FS. Inf A was detected in 16% of patients with simple FSs and 30% of patients with complex FSs and a significant difference between them (p &lt; 0.01). Conclusion The results of this study showed that respiratory viral and bacterial pathogens are important in the etiology of FS in children. It is considered that complex FSs may be triggered by Inf A. The fact is viral pathogens are very common; therefore, antibiotics must be carefully prescribed. These results also draw attention to the use of the quadrivalent influenza vaccine in the prevention of FS related to the flu.

Detection of Viral and Bacterial Respiratory Pathogens Identified by Molecular Methods in COVID-19 Hospitalized Patients and Its Impact on Mortality and Unfavorable Outcomes.

PurposeThe purpose of this study is to evaluate the frequency of viral and bacterial respiratory pathogens detected by molecular methods in sputum samples of patients hospitalized for COVID-19 and to evaluate its impact on mortality and unfavorable outcomes (in-hospital death or mechanical ventilation).Patients and MethodsThe prospective cohort included patients with diagnosis of COVID-19 hospitalized at Hospital Nacional Hipólito Unanue. Sociodemographic and clinical data were collected from clinical records. Sputum samples were analyzed with the Biofire Filmarray Pneumonia plus® respiratory panel. Crude and adjusted associations with unfavorable outcomes were evaluated using logistic regression models.ResultsNinety-three patients who were able to collect sputum samples were recruited between September 8 and December 28, 2020. The median age was 61.7 years (IQR 52.3–69-8) and 66 (71%) were male. The most frequent symptoms were dyspnea, cough, fever, and general malaise found in 80 (86%), 76 (82%), 45 (48%), and 34 (37%) patients, respectively. Fifty-three percent of patients had comorbidities. Seventy-six (82%) patients received antibiotics prior to admission and 29 (31%) developed unfavorable outcome. Coinfection was evidenced in 38 (40.86%) cases. The most frequently found bacteria were Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae and Klebsiella pneumoniae in 11 (11.83%), 10 (10.75%), 10 (10.75%), and 8 (8.6%) cases, respectively. Streptococcus pneumoniae was found in one case (1.08%). We neither identify atypical bacteria nor influenza virus. No association was found between the presence of viral or bacterial microorganisms and development of unfavorable outcomes (OR 1.63; 95% CI 0.45–5.82).ConclusionA high frequency of respiratory pathogens was detected by molecular methods in patients with COVID-19 pneumonia but were not associated with unfavorable outcomes. No atypical agents or influenza virus were found. The high use antibiotics before admission is a concern. Our data suggest that the use of drug therapy against atypical bacteria and viruses would not be justified in patients hospitalized for COVID-19.

Legionnaires’ disease in dental offices: Quantifying aerosol risks to dental workers and patients

Legionella pneumophila is an opportunistic bacterial respiratory pathogen that is one of the leading causes of drinking water outbreaks in the United States. Dental offices pose a potential risk for inhalation or aspiration of L. pneumophila due to the high surface area to volume ratio of dental unit water lines—a feature that is conducive to biofilm growth. This is coupled with the use of high-pressure water devices (e.g., ultrasonic scalers) that produce fine aerosols within the breathing zone. Prior research confirms that L. pneumophila occurs in dental unit water lines, but the associated human health risks have not been assessed. We aimed to: (1) synthesize the evidence for transmission and management of Legionnaires’ disease in dental offices; (2) create a quantitative modeling framework for predicting associated L. pneumophila infection risk; and (3) highlight influential parameters and research gaps requiring further study. We reviewed outbreaks, management guidance, and exposure studies and used these data to parameterize a quantitative microbial risk assessment (QMRA) model for L. pneumophila in dental applications. Probabilities of infection for dental hygienists and patients were assessed on a per-exposure and annual basis. We also assessed the impact of varying ventilation rates and the use of personal protective equipment (PPE). Following an instrument purge (i.e., flush) and with a ventilation rate of 1.2 air changes per hour, the median per-exposure probability of infection for dental hygienists and patients exceeded a 1-in-10,000 infection risk benchmark. Per-exposure risks for workers during a purge and annual risks for workers wearing N95 masks did not exceed the benchmark. Increasing air change rates in the treatment room from 1.2 to 10 would achieve an ∼85% risk reduction, while utilization of N95 respirators would reduce risks by ∼95%. The concentration of L. pneumophila in dental unit water lines was a dominant parameter in the model and driver of risk. Future risk assessment efforts and refinement of microbiological control protocols would benefit from expanded occurrence datasets for L. pneumophila in dental applications.

Design of a High-Throughput Real-Time PCR System for Detection of Bovine Respiratory and Enteric Pathogens.

Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves, Pasteurella multocida (38.4%), rotavirus A (27.4%), Mycoplasma spp. (26.2%), and Trueperella pyogenes (25.5%) were the most prevalent pathogens, while P. multocida (71.4%), Mycoplasma spp. (58.9%), Mannheimia haemolytica (53.6%), and Mycoplasma bovis (42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.

Bacterial and Viral Coinfections with the Human Respiratory Syncytial Virus.

The human respiratory syncytial virus (hRSV) is one of the leading causes of acute lower respiratory tract infections in children under five years old. Notably, hRSV infections can give way to pneumonia and predispose to other respiratory complications later in life, such as asthma. Even though the social and economic burden associated with hRSV infections is tremendous, there are no approved vaccines to date to prevent the disease caused by this pathogen. Recently, coinfections and superinfections have turned into an active field of study, and interactions between many viral and bacterial pathogens have been studied. hRSV is not an exception since polymicrobial infections involving this virus are common, especially when illness has evolved into pneumonia. Here, we review the epidemiology and recent findings regarding the main polymicrobial infections involving hRSV and several prevalent bacterial and viral respiratory pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, human rhinoviruses, influenza A virus, human metapneumovirus, and human parainfluenza viruses. As reports of most polymicrobial infections involving hRSV lack a molecular basis explaining the interaction between hRSV and these pathogens, we believe this review article can serve as a starting point to interesting and very much needed research in this area.

Extensive microbiological respiratory tract specimen characterization in critically ill COVID-19 patients.

Microbial co‐infections may contribute to the pulmonary deterioration in COVID‐19 patients needing intensive care treatment. The present study portrays the extent of co‐infections in COVID‐19 ICU patients. Conventional culture, molecular detections for atypical aetiologies, QiaStat‐Dx® respiratory panel V2 detecting 21 respiratory pathogens and ribosomal DNA genes 16S/18S amplicon‐based microbiome analyses were performed on respiratory samples from 34 COVID‐19 patients admitted to the ICU. Potential pathogens were detected in seven patients (21%) by culturing, in four patients (12%) by microbiome analysis and in one patient (3%) by respiratory panel. Among 20 patients receiving antibiotics prior to ICU admission, fungi (3 Candida albicans, 1 C. tropicalis, 1 C. dubliniensis) were cultured in 5 (15%) endotracheal aspirates. Among 14 patients who were antibiotic‐naive at ICU admission, two patients (6%) had bacterial respiratory pathogens (Staphylococcus aureus, Streptococcus pseudopneumoniae) cultured in their endotracheal aspirates. Microbiome analysis recognized four potential respiratory pathogens (3 Haemophilus influenza, 1 Fusobacterium necrophorum) isolated in samples from four other patients (12%). QiaStat‐Dx® respiratory panel V2 detected adenovirus in one patient (3%). The prevalence of pulmonary microbial co‐infections is modest among COVID‐19 patients upon admission to ICU. Microbiome analysis complements conventional microbial diagnostics in characterization of respiratory co‐infections.

A Screening of the MMV Pandemic Response Box Reveals Epetraborole as a New Potent Inhibitor against Mycobacterium abscessus.

Mycobacterium abscessus is the one of the most feared bacterial respiratory pathogens in the world. Unfortunately, there are many problems with the current M. abscessus therapies available. These problems include misdiagnoses, high drug resistance, poor long-term treatment outcomes, and high costs. Until now, there have only been a few new compounds or drug formulations which are active against M. abscessus, and these are present in preclinical and clinical development only. With that in mind, new and more powerful anti-M. abscessus medicines need to be discovered and developed. In this study, we conducted an in vitro-dual screen against M. abscessus rough (R) and smooth (S) variants using a Pandemic Response Box and identified epetraborole as a new effective candidate for M. abscessus therapy. For further validation, epetraborole showed significant activity against the growth of the M. abscessus wild-type strain, three subspecies, drug-resistant strains and clinical isolates in vitro, while also inhibiting the growth of M. abscessus that reside in macrophages without cytotoxicity. Furthermore, the in vivo efficacy of epetraborole in the zebrafish infection model was greater than that of tigecycline. Thus, we concluded that epetraborole is a potential anti-M. abscessus candidate in the M. abscessus drug search.

Co‐infection analysis of bacterial and viral respiratory pathogens from clinically healthy swine in Eastern China

Porcine respiratory disease complex (PRDC) is one of the most challenging health concerns for pig production worldwide. The aim of the present study was to determine the prevalence of pathogens associated with PRDC, including porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) and bacterial agents, such as Streptococcus suis, Haemophilus parasuis and Actinobacillus pleuropneumoniae, in clinically healthy pigs in Eastern China. Molecular detection revealed positive single‐pathogen detection rates of 59.9%, 27.2%, 52.3%, 33.2% and 0.4% for PCV2, PRRSV, S. suis, H. parasuis and A. pleuropneumoniae, respectively. Co‐infection with more than one pathogen was frequently detected in these samples, with PCV2/S. suis, H. parasuis and PCV2/H. parasuis mixed infection rates of 35.4%, 33.2% and 21.6%, respectively, and PCV2/S. suis/H. parasuis and PRRSV/PCV2/S. suis co‐infection rates of 21.6% and 6.2%, respectively. These results suggest that mixed infections are prevalent among PRDC cases in swine, which may pose a greater threat to the health of herds compared with single‐pathogen infections.

Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle.

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

Secondary diseases of infectious and non-infectious origin in patients diagnosed with COVID-19

The article considers the features of the occurrence and course of secondary diseases of infectious and non-infectious genesis in patients with a diagnosis of COVID-19, and also examines individual clinical cases described in the literature. The author concludes that the results showed that major respiratory diseases, in particular COPD (chronical obstructive pulmonary disease) and smoking, are associated with severe COVID-19 outcomes. The results of this study can help policy makers, doctors and healthcare professionals in the front line to make evidence-based decisions and reduce the mortality and morbidity of this 21st century pandemic. Understanding the proportion of COVID-19 patients with acute respiratory bacterial co-infection and pathogens is critical for the treatment of COVID-19 patients and helps ensure responsible use of antibiotics and minimize the negative effects of overuse. In addition, this knowledge may have a significant impact on clarifying recommendations for empirical antibiotic therapy for patients with COVID-19.

Influenza D virus in respiratory disease in Canadian, province of Québec, cattle: Relative importance and evidence of new reassortment between different clades

Influenza D virus (IDV), a segmented single-stranded negative-sense ribonucleic acid (RNA) virus, belongs to the new Delta influenza virus genus of the Orthomyxoviridae family. Cattle were proposed as the natural reservoir of IDV in which infection was associated with mild-to-moderate respiratory clinical signs (i.e. cough, nasal discharge and dyspnoea). In order to investigate the role of IDV in bovine respiratory disease, during the period 2017-2020, 883 nasal or naso-pharyngeal swabs from Canadian cattle with respiratory signs (cough and/or dyspnoea) were tested by (RT-)qPCR for IDV and other major bovine viral (bovine herpesvirus 1, bovine viral diarrhoea virus, bovine respiratory syncytial virus, bovine parainfluenza virus 3 and bovine coronavirus) and bacterial (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis) respiratory pathogens. In addition, whole-genome sequencing and phylogenetic analyses were carried out on five IDV-positive samples. The prevalence of IDV RT-qPCR (with cut-off: Cq<38) at animal level was estimated at 5.32% (95% confidence interval: 3.94-7.02). Positive result of IDV was significantly associated with (RT-)qPCR-positive results for bovine respiratory syncytial virus and Mycoplasma bovis. While phylogenetic analyses indicate that most segments belonged to clade D/660, reassortment between clades D/660 and D/OK were evidenced in four samples collected in 2018-2020. Relative importance of influenza D virus and associated pathogens in bovine respiratory disease of Canadian dairy cattle was established. Whole-genome sequencing demonstrated evidence of reassortment between clades D/660 and D/OK. Both these new pieces of information claim for more surveillance of IDV in cattle production worldwide.

Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens.

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

Exploration of Galectin Ligands Displayed on Gram-Negative Respiratory Bacterial Pathogens with Different Cell Surface Architectures.

Galectins bind various pathogens through recognition of distinct carbohydrate structures. In this work, we examined the binding of four human galectins to the Gram-negative bacteria Klebsiella pneumoniae (Kpn) and non-typeable Haemophilus influenzae (NTHi), which display different surface glycans. In particular, Kpn cells are covered by a polysaccharide capsule and display an O-chain-containing lipopolysaccharide (LPS), whereas NTHi is not capsulated and its LPS, termed lipooligosacccharide (LOS), does not contain O-chain. Binding assays to microarray-printed bacteria revealed that galectins-3, -4, and -8, but not galectin-1, bind to Kpn and NTHi cells, and confocal microscopy attested binding to bacterial cells in suspension. The three galectins bound to array-printed Kpn LPS. Moreover, analysis of galectin binding to mutant Kpn cells evidenced that the O-chain is the docking point for galectins on wild type Kpn. Galectins-3, -4, and -8 also bound the NTHi LOS. Microarray-assisted comparison of the binding to full-length and truncated LOSs, as well as to wild type and mutant cells, supported LOS involvement in galectin binding to NTHi. However, deletion of the entire LOS oligosaccharide chain actually increased binding to NTHi cells, indicating the availability of other ligands on the bacterial surface, as similarly inferred for Kpn cells devoid of both O-chain and capsule. Altogether, the results illustrate galectins’ versatility for recognizing different bacterial structures, and point out the occurrence of so far overlooked galectin ligands on bacterial surfaces.

A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry.

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.